High throughput chemical screening, of enzyme activity for example, typically involves quantitative detection of one or more substrate and/or product. The most universal detection method to date is mass spectrometry (MS), which allows identification of a particular organic molecule based on mass to charge ratio.
Traditionally, mass spectrometry is performed in tandem with liquid chromatography to purify and separate the components of interest. This purification can be considered to be on-line sequential purification. The sequential nature of the purification limits the ability of mass spectrometry to screen a large number of reaction products in a short amount of time, because the purification must occur in line with and previous to the mass spectrometry.
DNA shuffling technology is used to create a library of related gene sequences that encode, e.g., one or more enzyme that catalyzes a reaction. Such a library is constructed, e.g., by homologous exchange of DNA fragments during DNA shuffling.
In one typical set of embodiments, the library of related gene sequences is on a plasmid that has been transformed into a bacteria. Thus a single bacterial clone can carry a unique gene sequence representing a unique variant of a particular enzyme or enzyme pathway.
For directed evolution, the library is screened for variants having a desired characteristic. Evolution of enzymes and pathways involves biochemical reaction of one or more enzymes that can be detected by a chemical screening method. A chemical screening method detects the substrates and or products of the enzyme reaction(s).
Presently, the use of mass spectrometry to analyze these enzyme reactions is extremely time consuming. The time limitation is due to the need to separate and purify the products and reactants of the enzyme pathways before injection into a mass spectrometer. This limits the number of samples that can be analyzed to about 100 samples per day (typical purification runs (e.g., liquid chromatography) require about 10 minutes/sample. At 6 samples per hour, 144 samples can be run in a 24-hour period). A new high throughput system would be useful to provide a method of analyzing a library for a few mutants out of thousands that will provide the desired properties.
One recently developed system is the electrospray method as described in “Quantitative Electrospray Mass-Spectrometry for the Rapid Assay of Enzyme Inhibitors,” by Wu et al. in Chemistry & Biology 1997, Vol. 4 No. 9, p653–657. Electrospray ionization is a mild method of transferring charged polar organic molecules into the gas phase for mass spectrometry analysis and is applicable for most biologically relevant organic molecules. The electrospray method eliminates the need for prior derivatization of samples before injection into a mass-spectrometer as in GC/MS and thus shortens the analysis time for mass spectrometry. However, column separation is still utilized in this technique, limiting throughput as noted above.
Another recent development is described in “Fast Screening for Drugs of Abuse by Solid-Phase Extraction Combined with Flow-Injection Ionspray-Tandem Mass Spectrometry,” by Weinman and Svobodain, Journal of Analytical Toxicology, Vol. 22, 1998, p. 319–328. The technique described combined tandem mass spectrometry and electrospray methods to simultaneously detect different drugs in serum or urine. Although no column separation was used because the tandem mass spectrometry allowed detection of multiple compounds, a solid phase extraction method was necessary in the sample preparation. The sample preparation steps were still too lengthy to provide high throughput screening by mass spectrometry.
Accordingly, a high throughput method of performing mass spectrometry, e.g., for screening libraries of shuffled molecules, would be useful. The present invention fulfills these and many other needs which will become apparent upon complete review of this disclosure.